A simple and efficient MEKC method was developed to simultaneously determine human insulin, its five analogues, the main degradation products and the excipients usually present in injection formulations ... [more ▼]

A simple and efficient MEKC method was developed to simultaneously determine human insulin, its five analogues, the main degradation products and the excipients usually present in injection formulations. A very fast method with a total analysis time of 3 min was then successfully validated for the analysis of human insulin and the quality control of different commercial formulations was carried out. [less ▲]

Counterfeiting is a widespread problem in the world. The medicines, like insulin or GHRP, need a strict quality control. Capillary electrophoresis and liquid chromatography methods were developed to ... [more ▼]

Counterfeiting is a widespread problem in the world. The medicines, like insulin or GHRP, need a strict quality control. Capillary electrophoresis and liquid chromatography methods were developed to analyze these peptides. The human insulin and its different analogues (lispro, aspart, glulisin, glargin and detemir) were separated by MEKC within 15 minutes. The GHRP-2 and -6 were separated by HPLC also in 15 minutes. Several samples of GHRP-6 were analyzed and non-compliances were reported. These analytical approaches seem to be promising to fight against the counterfeiting of such medicines. [less ▲]

Erectile dysfunction drugs are among the most counterfeit drug classes in industrialized countries. To fight against this plague, several analytical approaches are available for control laboratories. The ... [more ▼]

Erectile dysfunction drugs are among the most counterfeit drug classes in industrialized countries. To fight against this plague, several analytical approaches are available for control laboratories. The present article reviews the main used techniques and concludes presenting a general strategy for the detection and handling of drug counterfeits. [less ▲]

Insulin plays an important role in the homeostasis of blood glucose concentration. A deficiency of this hormone causes diabetes, which can be treated by subcutaneous injection of synthetic insulin ... [more ▼]

Insulin plays an important role in the homeostasis of blood glucose concentration. A deficiency of this hormone causes diabetes, which can be treated by subcutaneous injection of synthetic insulin. Besides human regular insulin, several modified analogues have been developed to accelerate (Lispro, Aspart, Glulisin) or delay (Glargin, Detemir) its absorption. Moreover, protamine is sometimes associated with human, Lispro or Aspart insulin to give a crystalline form, which delays the action of insulin, providing it with a prolonged absorption profile after injection. Diabetes is one of the most common metabolic diseases in the world; its prevalence increases continuously. A lot of patients are therefore concerned with the treatment, which is relatively expensive and requires a prescription. Some pharmaceutical formulations can sometimes be found without prescription on the parallel market but the risk of drug counterfeiting is then considerably increased. The poor quality of these drugs can lead to harmful consequences for the public health. It is therefore essential to develop a suitable method for the identification and quantification of human insulin and its analogues inside formulations. Ortner et al. [1] have already proposed micellar electrokinetic chromatography (MEKC) methods to detect simultaneously human insulin and its five analogues but no quantitative applications were presented. Furthermore, formulations containing protamine were not tested so we included them in our study. The first optimisation step involved the sample preparation procedure. An acidic sample solution (10 mM HCl) was finally selected to solubilise protamine and Glargin. Then the background electrolyte composition was investigated to separate the components present in the formulations. A basic buffer (50 mM ammonium acetate pH 9) was selected, providing an important and stable electroosmotic flow, a negative charge to the insulins and avoiding any adsorption to the capillary wall. The addition of sodium dodecylsulfate (SDS) and acetonitrile (ACN) was also found crucial for selectivity. With 50 mM SDS and 15% ACN the six insulins and the two major excipients (phenol and meta-cresol) were fully separated within 15 minutes. This method was then entirely validated for the human insulin and the quality control of related formulations was performed. The next step will be the validation and the quantification of the other analogues. [less ▲]

The ophthalmic solution of voriconazole, i.e. (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoropyrimidin-4-yl)-1-(1H-1,2,4-triazol-1-y l)butan-2-ol, made from an injection formulation which also contains ... [more ▼]

The ophthalmic solution of voriconazole, i.e. (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoropyrimidin-4-yl)-1-(1H-1,2,4-triazol-1-y l)butan-2-ol, made from an injection formulation which also contains sulfobutylether-beta-cyclodextrin sodium salt as an excipient (Vfend((R))), is used for the treatment of fungal keratitis. A liquid chromatographic (LC) method using polar organic mobile phase and cellulose tris(4-chloro-3-methylphenylcarbamate) coated on silica as chiral stationary phase was successfully developed to evaluate the chiral stability of the ophthalmic solution. The percentage of methanol (MeOH) in the mobile phase containing acetonitrile (ACN) as the main solvent significantly influenced the retention and resolution of voriconazole and its enantiomer ((2S,3R)-2-(2,4-difluorophenyl)-3-(5-fluoropyrimidin-4-yl)-1-(1H-1,2,4-triazol-1- yl)butan-2-ol). The optimized mobile phase consisted of ACN/MeOH/diethylamine/trifluoroacetic acid (80/20/0.1/0.1; v/v/v/v). The method was found to be selective not only regarding the enantiomer of voriconazole but also regarding the specified impurities described in the monograph from the European Pharmacopoeia. The LC method was then fully validated applying the strategy based on total measurement error and accuracy profiles. Under the selected conditions, the determination of 0.1% of voriconazole enantiomer could be performed. Finally, a stability study of the ophthalmic solution was conducted using the validated LC method. [less ▲]

An original micellar electrokinetic chromatography (MEKC) method using in-capillary derivatization with a chiral labeling reagent was developed for the separation of amino acid (AA) derivatives. The ... [more ▼]

An original micellar electrokinetic chromatography (MEKC) method using in-capillary derivatization with a chiral labeling reagent was developed for the separation of amino acid (AA) derivatives. The potential of (-)-1-(9-fluorenyl)-ethyl chloroformate (FLEC) as in-capillary derivatization agent is described for the first time. Several parameters for in-capillary derivatization and subsequent MEKC separation were systematically investigated using experimental designs. Firstly experimental conditions for in-capillary derivatization were optimized using face-centered central composite design (FCCD). Mixing voltage and time as well as concentration of the labeling solution were investigated. Efficient labeling was achieved by sequential injection of AAs and FLEC labeling solution followed by the application of a voltage of 0.2kV for 570s. The background electrolyte (BGE) composition was then optimized in order to achieve selectivity. A FCCD was performed with two factors, namely the sodium dodecyl sulfate (SDS) concentration and the percentage of propan-2-ol (IPA). The separation of 12 pairs of derivatized AA (FLEC-AA) diastereomers was achieved with resolution values comprised between 3 and 20. Furthermore, an efficient derivatization and separation of 29 FLEC-AA derivatives were achieved in a single run using a buffer made up of 40mM sodium tetraborate, 21mM SDS and 8.5% IPA. The method was successfully applied to the analysis of spiked artificial cerebrospinal fluid (aCSF) sample. [less ▲]

In the course of our investigations on Umutambasha in order to identify its convulsant principles, small quantities of monofluoroacetate were observed in stem bark, leaves, and fruits of this plant newly ... [more ▼]

In the course of our investigations on Umutambasha in order to identify its convulsant principles, small quantities of monofluoroacetate were observed in stem bark, leaves, and fruits of this plant newly identified as Dichapetalum michelsonii Hauman. Conclusive evidence for a monofluoroacetate presence came from its isolation from the freeze-dried extract of stem bark. Three free unusual amino acids, named N-methyl-α-alanine, N-methyl-β-alanine, and 2,7-diaminooctan-1,8-dioic acid, described for the first time in a plant, and known trigonelline were also isolated from the stem bark of D. michelsonii. Structure elucidations were mainly achieved by spectroscopic methods (1H-NMR, 2D-NMR, MS) and by comparison with authentic references. These unusual amino acids were detected by a fast, reliable TLC analysis in all our batches of Umutambasha, suggesting that they could be used for identification purposes in case of human or livestock intoxications. Finally, EEG recordings and behavioural observations performed in mice suggested that the convulsive patterns produced by Umutambasha are the consequence of monofluoroacetate presence in D. michelsonii. [less ▲]

Insulin plays an important role in the homeostasis of blood glucose concentration. A deficiency of this hormone causes diabetes, which can be treated by subcutaneous injection of synthetic insulin ... [more ▼]

Insulin plays an important role in the homeostasis of blood glucose concentration. A deficiency of this hormone causes diabetes, which can be treated by subcutaneous injection of synthetic insulin. Besides human regular insulin, several modified analogues have been developed to accelerate (Lispro, Aspart, Glulisin) or delay (Glargin, Detemir) its absorption. Moreover, protamine is sometimes associated with human, Lispro or Aspart insulin to give a crystalline form, which delays the action of insulin, providing it with a prolonged absorption profile after injection. Diabetes is one of the most common metabolic diseases in the world; its prevalence increases continuously. A lot of patients are therefore concerned with the treatment, which is relatively expensive and requires a prescription. Some pharmaceutical formulations can sometimes be found without prescription on the parallel market but the risk of drug counterfeiting is then considerably increased. The poor quality of these drugs can lead to harmful consequences for the public health. It is therefore essential to develop a suitable method for the identification and quantification of human insulin and its analogues. Ortner et al. have already proposed micellar electrokinetic chromatography (MEKC) methods to analyse simultaneously human insulin and its five analogues but formulations containing protamine were not tested. Since the number of these formulations is significant, we included them in our study. The first optimisation step involved the sample preparation procedure. An acidic sample solution (0.01 M HCl) was finally selected to solubilise protamine and Glargin. Then the background electrolyte composition was investigated to separate the components present in the formulations. A basic buffer (50 mM ammonium acetate pH 9) was selected, providing an important and stable electroosmotic flow, a negative charge to insulin and related compounds and avoiding any adsorption to the capillary wall. The addition of sodium dodecyl sulfate and acetonitrile were also found crucial for selectivity. The six insulins and the two major excipients (phenol and m-cresol) were fully separated within 15 minutes The aforementioned method was then adapted to permit the separation of each insulin from its degradation products. [less ▲]

Background: We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this ... [more ▼]

Background: We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. Methodology/Principal Findings: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. Conclusions and General Significance: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling. [less ▲]

Since antimalarial drugs counterfeiting is dramatically present on the African market, the development of simple analytical methods for their quality control is of great importance. This work consists in ... [more ▼]

Since antimalarial drugs counterfeiting is dramatically present on the African market, the development of simple analytical methods for their quality control is of great importance. This work consists in the CE analysis of 15 antimalarials (artesunate, artemether, amodiaquine, chloroquine, piperaquine, primaquine, quinine, cinchonine, mefloquine, halofantrine, sulfadoxine, sulfalen, atovaquone, proguanil, and pyrimethamine). Since all these molecules cannot be ionized at the same pH, MEKC was preferred because it also allows separation of neutral compounds. Preliminary experiments were first carried out to select the most crucial factors affecting the antimalarials separation. Several conditions were tested and four parameters as well as their investigation domain were chosen: pH (5–10), SDS concentration (20–90 mM), ACN proportion (10–40%), and temperature (20–35°C). Then, the experimental design methodology was used and a central composite design was selected. Mathematical modeling of the migration times allowed the prediction of optimal conditions (29°C, pH 6.6, 29 mM SDS, 36% ACN) regarding analyte separation. The prediction at this optimum was verified experimentally and led to the separation of 13 compounds within 8 min. Finally, the method was successfully applied to the quality control of African antimalarial medicines for their qualitative and quantitative content. [less ▲]

In 1984, Terabe and co-workers introduced a modified version of CZE, called micellar electrokinetic chromatography (MEKC), in which surfactant-formed micelles are included in the running buffer providing ... [more ▼]

In 1984, Terabe and co-workers introduced a modified version of CZE, called micellar electrokinetic chromatography (MEKC), in which surfactant-formed micelles are included in the running buffer providing a two phases chromatographic system for the separation of neutral compounds. Depending on their hydrophobicity, compounds can interact with the core of the micelles. Many MEKC methods have been described for the separation of neutral and basic compounds and for various applications. However, the migration behaviour of cationic, anionic and neutral analytes mixture is not well established regarding to the surfactant concentration and to the proportion of organic solvent in the background electrolyte (BGE). With such a mixture, it is important to remember that the separation of charged solutes in MEKC involves a combination of chromatographic and electrophoretic separation mechanisms. Moreover, it is worth noting that with charged analytes, two kinds of interactions with the micelles may occur; not only with the hydrophobic core but also with the head groups of the micelles through electrostatic interactions. In systems in which the surfactant has an opposite charge to that of the solute, ion pairing may occur. On the contrary, when a solute and the surfactant have similar charges, coulombic forces may cause the repulsion of these molecules. The goal of this study is to improve our understanding of the migration behaviour of charged and uncharged analytes in MEKC systems. With this aim in view, effective mobility (electrophoretic mobility under the influence of micelles) of neutrals, cations and anions were mesured at neutral, basic and acidic pH with BGE containing different SDS concentrations and ACN proportions. This study is also focused on the changes of migration order between CZE and MEKC systems using different BGE compositions. In the MEKC systems investigated in the study, SDS concentration and ACN proportion show a tremendous effect on the effective mobilities and migration order of the model compounds. While anions interact very weakly with SDS micelles, neutrals and cations interact with SDS through hydrophobic and ionic bonds. These interactions become stronger with the increase of SDS concentration and weaker with high ACN proportion. With 20 mM of SDS in the BGE, CZE behaviour is observed till 40% of ACN. But, when the SDS concentration is high and the ACN proportion is low, the migration order of analytes is reversed compared to CZE: EOF first, then the anions, followed by the neutrals and finally the cations. The migration order inside each group (cations, neutrals and anions) depends on the hydrophobicity of the analytes. Different organic solvents were also investigated to study the ion-pair formation. Those observations confirm the interest of using MEKC not only for the separation of neutral compounds but also variously charged analytes. [less ▲]

The migration behaviour of charged and uncharged analytes in MEKC was investigated under different conditions. Effective mobilities – electrophoretic mobilities under the influence of the negatively ... [more ▼]

The migration behaviour of charged and uncharged analytes in MEKC was investigated under different conditions. Effective mobilities – electrophoretic mobilities under the influence of the negatively charged micelles – of cations, anions and neutrals were measured at neutral, basic and acidic pH values (7.5, 11 and 2.2) using background electrolytes containing different sodium dodecyl sulfate (SDS) concentrations (0-90 mM) and acetonitrile proportions (0-75 %, v/v). The SDS concentration and acetonitrile proportion were found to have a tremendous effect on the effective mobilities and the migration order of the tested compounds. Although the SDS micelles interact more strongly with neutrals and cations, the migration of anionic compounds is also affected by the SDS concentration, indicating that hydrophobic interactions can occur between the micelles and these compounds. Since cationic, anionic and neutral solutes exhibit rather different migration behaviours, it is possible to considerably enhance the separation selectivity by properly adjusting the SDS concentration and the acetonitrile proportion in the background electrolyte. These observations confirm the interest of using MEKC not only for the separation of neutral substances but also for the analysis of mixtures of charged compounds. [less ▲]

In this study, the migration behavior of charged and uncharged analytes was investigated under different conditions. Effective mobilities - electrophoretic mobilities under the influence of micelles - of ... [more ▼]

In this study, the migration behavior of charged and uncharged analytes was investigated under different conditions. Effective mobilities - electrophoretic mobilities under the influence of micelles - of cations, anions, and neutrals were measured at neutral, basic, and acidic pH (7.5, 11, and 2.2) using background electrolytes containing different sodium dodecyl sulfate (SDS) concentrations (0-90 mM) and acetonitrile (ACN) proportions (0-75%). SDS concentration and ACN proportion were found to have a tremendous effect on the effective mobilities and migration order of the model compounds. Although the SDS micelles preferably interact with neutrals and cations, hydrophobic bonds can also occur with anions. Cations, anions, and neutrals having rather different migration behaviors, it is possible to considerably enhance the selectivity of the method by adjusting properly the SDS concentration and the ACN proportion. These observations confirm the interest of using micellar electrokinetic chromatography not only for the separation of neutral substances but also to analyze charged compounds. [less ▲]

A novel dual chiral CE method was developed for the separation of l- and d-amino acids (AAs), using in-capillary derivatization with 9-fluoroenylmethyl chloroformate (FMOC). Firstly, using pre-column ... [more ▼]

A novel dual chiral CE method was developed for the separation of l- and d-amino acids (AAs), using in-capillary derivatization with 9-fluoroenylmethyl chloroformate (FMOC). Firstly, using pre-column derivatization, the enantioseparation of FMOC-AAs was optimized according to the nature of cyclodextrins (CD). A background electrolyte (BGE) composed of 30mM beta-CD, 30mM octakis(2,3-dihydroxy-6-O-sulfo)-gamma-CD (OS-gamma-CD), 40mM tetraborate and 15% isopropanol (IPA) was selected and led to 17 baseline resolved pairs (R(s)=1.7-5.8) and two partially resolved pairs (Lys, R(s)=0.5 and Arg, R(s)=1.2). Experimental conditions for in-capillary derivatization were then optimized. Several parameters, such as mixing voltage and time, concentration of labeling solution and the length of the spacer plug were studied. The optimal conditions for in-capillary derivatization procedure were obtained using successive hydrodynamic injections (30mbar) of AAs for 2s, borate buffer for 4s and 10mM FMOC solution for 6s, followed by a mixing at 3kV for 72s and wait time of 1min. Moreover, a particular attention was paid to improve separation chemoselectivity. The effect on stereoselectivity and chemoselectivity of different factors, such as decrease of pH and tetraborate concentration and the addition of sodium dodecyl sulfate (SDS), was investigated using the in-capillary derivatization procedure. The best separation of a standard mixture of ten AA racemates was observed using a BGE containing 30mM beta-CD, 30mM OS-gamma-CD, 25mM SDS, 40mM sodium tetraborate and 17% IPA. [less ▲]